Two novel members of Onygenales, Keratinophyton kautmanovae and K. keniense spp. nov. from soil

Two new Keratinophyton species, K. kautmanovae sp. nov. and K. keniense sp. nov., isolated from soil samples originating from two different geographical and environmental locations (Africa and Europe) are described and illustrated. Phylogenetically informative sequences obtained from the internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) rDNA, as well as their unique phenotype, fully support novelty of these two fungi for this genus. Based on ITS and LSU combined phylogeny, both taxa are resolved in a cluster with eight accepted species, including K. alvearium, K. chongqingense, K. hubeiense, K. durum, K. lemmensii, K. siglerae, K. submersum, and K. sichuanense. The new taxon, K. kautmanovae, is characterized by clavate, smooth to coarsely verrucose conidia, absence of arthroconidia, slow growth at 25 °C, and no growth at 30 °C, while K. keniense is morphologically unique with a high diversity of conidial shapes (clavate, filiform, globose, cymbiform and rhomboid). Both species are described based on their asexual, a chrysosporium-like morph. While the majority of hitherto described Keratinophyton taxa came from Europe, India and China, the new species K. keniense represents the first reported taxonomic novelty for this genus from Africa.

The genus Keratinophyton is represented by a large group of keratinolytic soil-borne fungi rather common in areas with high animal activity resulting in transfer of keratinous material to the soil.Ecology and distribution of the genus has been reviewed in a previous study, stating that soil and soil-like substrata are primary habitats for this fascinating group of onygenalean fungi 7 .Currently, this genus comprises a total of 27 recognized and accepted taxa 6,8 .
We performed a mycological survey with emphasis on keratinophilic fungi in environmental samples taken from Republic of Kenya, Africa and Slovak Republic, Europe.Herein, we present description of two novel morphologically and phylogenetically distant species within the genus Keratinophyton being characterized by their unique chrysosporium-like morphs.

Sample collection and isolation of the fungi
The soil sample was collected in front of the Budúcnosť adit at the abandoned antimony (Sb) deposit Pezinok-Kolársky Vrch, 48°19′03.3"N 17°14′20.9"E, locality Rudné Mines, Pezinok (Slovak Republic) in December 2021.Sampling site was situated approximately one meter from neutral mine drainage contaminated by potentially toxic elements.
The samples from the surface layer (up to 20 cm deep) were dried and stored in at 5-8 °C.Isolation of the keratinophilic fungi was performed as previously described 15 , with a modification according to 7 .A sample was divided into five subsamples.The subsamples (5 g each) were poured into Petri dishes (50 mm in diameter) and mixed with 0.5 g Vermiculite, then soaked with 3-4 mL (depending on moiety of the sample) antibiotic solution containing 0.5 g/L cycloheximide and 0.1 g/L chloramphenicol.Sterile defatted horse hair fragments (10 pieces of ca 2.0 cm per plate) were used as baits.The Petri dishes were then incubated at laboratory temperature (23-25 ± 1 °C), in dark, for a period of 2-3 months and remoistened with sterile deionized water when necessary 7,15 .The Petri dishes were checked weekly for the presence of fungi, and colonies were transferred on Sabouraud 4% dextrose agar (SDA; Merck, Darmstadt, Germany) supplemented with 0.5 g cycloheximide and 0.05 g chloramphenicol.Pure cultures were then transferred onto potato dextrose agar (PDA; Van Waters and Rogers (VWR) International, Leuven, Belgium) 7 .

Morphological analysis
The preliminary identification of the resulting keratinophilic fungi was carried out based on their phenotypic characteristics [9][10][11] .
For phenotypic determination, the strains were transferred by three-point inoculation onto PDA, Malt Extract Agar (MEA; Merck, Darmstadt, Germany), and SDA, and incubated for 14 d in the dark at 25 °C.Christensen´s urea agar (Sigma-Aldrich, St Louis, MO, USA) was used for additional physiological and biochemical characteristics (25 °C, 14 days, in the dark) 7 .
Colony growth rate (mm), colony structure and characteristics such as production of exudates and pigments were noted after 14 days (on PDA, MEA, and SDA).However, the cultivation was extended up to 3 months to observe and record changes in pigmentation of the colonies as well as to determine the onset of sexual reproduction 7 .In order to determine the optimal and minimum/maximum temperatures for growth, PDA, MEA and SDA plates were incubated at 5 °C, 8 °C, 10 °C, 12 °C, 15 °C, 18 °C, 20 °C, 25 °C, 28-32 °C, 35 °C, and 37 °C, and the growth rate was measured on the 14th day of cultivation.For comparative descriptions of the macroscopic and microscopic characteristics, PDA was used according to 7,11,16 .
For observation of microscopic traits 14-18 days growth on PDA was used.Conidiophore and conidia formation were observed in situ under low magnification (50-100×).Details of conidiophores, conidia (aleurioconidia) and other microscopic structures, such as width of hyphae, were observed in Melzer´s reagent and lactic acid with cotton blue 7 .Photomicrographs were taken using phase and Nomarski contrast optics on an Olympus BX51 microscope with Olympus DP72 camera and QuickPHOTO Micro 3.0 software.Photographs of the colonies were taken with a Sony DSC-RX100.
Dried fungal specimens were deposited as holotypes in the collections of the Mycological Department, National Museum in Prague, Czech Republic (PRM); ex-type cultures were deposited in the Bioactive Microbial Metabolites (BiMM) Fungal Collection, UFT-Tulln in Austria and in the Culture Collection of Fungi in Prague (CCF) 7 .

Keratinolytic activity
A hair perforation test was performed following de Hoog et al. (2020) using 25 mL water containing 2-3 drops 10% yeast extract (YEW) 17 .The hairs were examined microscopically 14 and 21 days after inoculation at 25 °C in the dark.At the end of the incubation period, a few pieces of hair were taken out from the testing medium.The overgrowing fungus was deactivated with 70% ethanol and then removed from the hair surface mechanically in a stream of a tap water 7 .The degree of hair digestion-degradation (keratinolytic activity) was assessed in the light microscope under 100× and 400× magnification.Water was used as mounting fluid for the observation and microphotography of the hair samples.Intensity of degradation 18 of hair was estimated on a scale of 0 to 4: 0 = no degradation; 0-1 = light degradation on the cuticle; 1 = moderate degradation on the cuticle and/or rare formation of boring hyphae; 2 = degradation of cuticle and cortex, with about 20% degradation; 3 = degradation of cuticle and cortex, with about 50% degradation; 4 = degradation of cuticle and cortex, with about 80% degradation.

DNA extraction, PCR amplification and sequencing
DNA was extracted using a standard cetyltrimethyl ammonium bromide (CTAB) procedure, as described previously 7,19 .The internal transcribed spacer (ITS) region was amplified with primers ITS1-F 20 and ITS4 21 using Taq-polymerase (GoTaq G2 Green Master Mix from Promega).The D1/D2 domains of the large-subunit (28S) rDNA gene (LSU) were amplified and sequenced using the primer pair ITS1/TW14 21,22 .All reactions were performed in an Eppendorf Gradient MasterCycler (Eppendorf, Hamburg, Germany).Conditions for amplification of ITS and LSU domains: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 90 s, and finally 5 min at 72 °C7 .The PCR products were sequenced with the same primers used for the PCR amplifications (LGC, Berlin, Germany).All sequences obtained in this study were deposited in GenBank nucleotide database (Table 1).

Phylogenetic analysis
All sequences were aligned with MAFFT v7 with default settings.The percent similarity between strains was determined using BioEdit v7.2 23 .ModelFinder 24 on IQ-TREE web server 25 was used to find the best-fitting model for ITS and LSU datasets according to the Bayesian Information Criterion (BIC).Phylogenetic trees were constructed using the maximum likelihood (ML) methods implemented in IQ-TREE web server.Branch support values were measured using ultrafast bootstraps.Additionally, MrBayes v3.2.7 26 with default settings on the CIPRES portal (http:// www.phylo.org/) was used for both datasets.Ctenomyces serratus CBS 187.61 (Arthrodermataceae, Onygenales) was used as an outgroup.Phylogenetic trees were displayed and edited using Treeview v1.6.6 27 and iTOL v6 28 .

Morphological analyses and keratin degradation
The results of the morphological analyses are given for each novel species under the Taxonomy section below.Temperature dependent growth of the new Keratinophyton species on PDA, MEA and SDA after 14 days are provided in Table S1a-c.Briefly, K. keniense grew better than K. kautmanovae on the same type of media and at the same incubation temperatures.All species showed good growth at 20-25 °C on all three media.K. kautmanovae does not grow at 30 °C, while K. keniense reached up to 38 mm after 14 days at this temperature.
Ability to digest keratin was observed in the two new species after 21 days on testing medium (YEW).However, attack intensity on the hair according to the scale of 18 Marchisio et al. was detected to be very weak in both species (= 0-1).Urease activity was negative in both new species on Christensen ´s urea agar.

Phylogenetical analysis
The ITS dataset consisted of 48 strains with 541 sites, LSU dataset consisted of 38 strains with 564 sites, and the ITS-LSU combined dataset included 38 strains with 564 sites.The best-fitting model was TNe + I + G4 for both datasets.Phylogenetic analyses of ITS (Fig. 1a) and combination of ITS-LSU data (Fig. 1b) of the species described in Keratinophyton and Aphanoascus revealed that strains BiMM-F297 and BiMM-296 (Keratinophyton kautmanovae sp.nov.) formed a basal clade in the genus Keratinophyton with 100% support.Strain BiMM-F335 (Keratinophyton keniense sp.nov.) was clustered together with K. hubeiense and K. sichuanense with 94% and 96% ITS similarity, respectively.
(Figs. 2 and 3) MycoBank: MB851666 Etymology: Named in honour of Ivona Kautmanová, Department of Botany, Slovak National Museum-Natural History Museum, Bratislava, Slovak Republic, an expert in the fungal ecology and taxonomy of higher fungi.
Culture characteristics: Colonies on PDA 13-15 mm diam at 25 °C, after 14 d, floccose to downy (mealy), with abundant sporulation, white to creamy, flat, slightly umbonate at the centre, with slightly radial colony margin submersed into agar, reverse yellowish with dark yellow centre, no pigment or exudate produced.At 30 °C, no growth (germination only).Colonies on SDA 7-10 mm diam at 25 °C, after 14 days, morphology similar to when on PDA with more floccose and plane colony surface and radial colony margin, poor sporulation, with orange reverse.At 30 °C, no growth (no germination).Colonies on MEA 15-20 mm diam at 25 °C, after 14 days, morphology similar to PDA with more floccose colonies and plane structure, with moderate sporulation, limited hyaline exudate present, with yellow-orange reverse and vivid orange centre.At  Optimum temperature for growth on PDA, SDA and MEA at 20-25 °C.Minimum growth (2-5 mm in diam) at 12 °C.Germination of the conidia observed at 10 °C.Maximum temperature for growth (1-3 mm in diam) at 28 °C.Keratinolytic activity very weak, with hair attack intensity = 0-1.Urease activity negative (after 20 days of incubation).
Diagnosis: Keratinophyton kautmanovae molecularly can be distinguished from other Keratinophyton species by ITS locus analysis.Combination of the following phenotypic features can be used to differentiate this fungus from other species in the genus: (1) obovoid-clavate, smooth to coarsely verrucose or irregularly warty conidia, (2) absence of arthroconidia, (3) generally slow grow at 25 °C, poor growth at 28 °C and no growth at 30 °C, and (4) orange reverse at 25 °C on MEA and SDA.
Notes: Based on a search of NCBI GenBank nucleotide database, the closest hit for Keratinophyton kautmanovae using the ITS sequence is Keratinophyton lemmensii (ex-type CCF 6359 = BiMM-F 76; GenBank   Optimum temperature for growth on PDA, SDA and MEA at 20-30 °C.Minimum growth (2-6 mm in diam) at 10 °C.Germination of the conidia observed at 8 °C.Maximum temperature for growth (2-5 mm in diam) at 33 °C.Germination of the conidia and formation of microcolonies observed at 34 °C.Keratinolytic activity absent, with hair attack intensity = 0. Urease activity negative (after 20 days of incubation).
Diagnosis: Keratinophyton keniense molecularly can be distinguished from other Keratinophyton species by ITS locus analysis.Combination of the following phenotypic features can be used to differentiate this fungus from other species in the genus: (1) cymbiform and rhomboid conidia, (2) arthroconidia very rare, (3) good grow at 20-30 °C, (4) dull yellow reverse at 25 °C.

Phylogeny
Phylogenetic reconstruction using ITS and LSU sequences (Fig. 1) resulted in clustering the both new taxa, Keratinophyton kautmanovae and K. keniense, with eight currently accepted species, namely K. alvearium 14 , K. durum 2 , K. chongqingense 8 , K. hubeiense 12 , K. lemmensii 7 , K. sichuanense 8 , K. siglerae 29 , and K. submersum 11 .Apart from the pronounced differences in the ITS regions, the species mentioned above can be distinguished by particular combinations of their phenotypic traits (e.g., colony characteristics, and morphology of conidia) as listed in Table 2.The monophyletic genus Keratinophyton is now extended and includes 29 species including six species known from sexual morphs 5 and 23 species which are currently known only from asexual morphs 8 and this study.The ability to produce ascosporic state (sexual morph) in vitro within this cluster is confined to K. durum 2 , characterized by discoid ascospores with flattened poles and with a broad equatorial rim, cruciform in side view, broad-ridged, with reticulate surface 4 .As noted by Li et al. (2022)  8 , the ability to form sexual morph in vitro is not phylogenetically conserved, as it can be seen from the phylogenetic analysis, showing that all known species forming ascosporic (sexual) structures within the genus are not clustered together and they are spread over the phylogenetic tree [6][7][8] .

Ecology and distribution
Almost all known Keratinophyton species have been isolated from soil or soil-like substrates, such as river sediments, compost and sand, and as non-pathogenic fungi as a result of mycological screening for so-called keratinophilic/keratinolytic fungi using a horse-hair baiting method 7,8 .This highly selective method was introduced by Vanbreuseghem (1952) for soil fungi having affinity to keratinous material especially for onygenalean fungi such as dermatophytes 19,30 .According to Papini et al. (1998), Ajello reviewed the taxonomy of keratinophilic fungi for the first time in 1968 31 .Later, Otčenášek et al. (1969) reported on the worldwide distribution of keratinophilic mycobiota in soil, claiming that the occurrence of keratinophilic fungi in soil depends on the presence of mammals, birds, and humans in a variety of ecological sites 32 .It is in fact the only method how this group of keratinophilic fungi can be isolated from the soil-like substrates and studied further.In this study, both www.nature.com/scientificreports/fungi originated from the areas which are freely accessible by wild animals typically inhabiting these regions, and thus, these soils might be presumably reach on source of keratin as well as a source of biodiversity for this specific fungal group regardless of geochemical properties of soils 15,33,34 .As for the sample from the abandoned antimony (Sb) deposit (Slovakia, EU), the soil sample is rich in iron oxides and is also characterized by elevated concentrations of arsenic, antimony, aluminum and sulfates.More about mineralogy and geochemistry of the studied site was published previously 35 .On the other hand, the soils in Egerton Njoro area are Vintric Mollic andosols 36,37 .The sampled area is native, not influenced by agricultural or any industrial activities.
As mentioned above, the genus Keratinophyton harbours a total of 29 species, including the two new species described in this study.The majority of the new taxa have been so far described from Europe (15 spp.),  followed by Asian continent (China, 8 spp.and India, 5 spp.) [5][6][7][8] .To the best to our knowledge, the description of K. keniense represents the first taxonomical novelty of the genus Keratinophyton from African continent.Thus, further research is needed because unknown strains may be isolated from similar environments on the African continent.
Hubálek provided a list of keratinolytic fungi associated with free-living mammals and birds of which ubiquitous K. durum, K. pannicola and K. terreum have been isolated from a variety of animals and from different geographical regions 38 .There are only a few reports of a human or animal clinical isolate belonging to Keratinophyton 16,39 , however, all these cases have doubtful etiological history and with no solid evidence of their pathogenicity 16,40 .On the other side, K. pannicola (as C. pannicola) is included in the Atlas of Clinical Fungi 17 as a concern in skin infections.Even though the keratinophilic fungi were considered as potential pathogens by several researchers 41,42 ; they rarely cause infections.Therefore, soil is proposed as an epidemiological and probably also an evolutionary link, that relates geophilic, zoophilic, and anthropophilic keratinophilic fungi 42 .Although being cycloheximide resistant, a potential pathogenicity to homeothermic vertebrates (mammals and birds) by these fungi seems highly unlikely because both new species are not able to grow at higher temperature (above 30-34 °C), they are urease negative, and possess none or very mild keratinolytic activity in vitro.Rather contrary, these fungi might be interesting from a metabolic point of view, as they undoubtedly represent a yet unexplored source of new bioactive compounds as there is not much known of these properties in the genus 34 .Metabolic profile and investigation of the potential use of substances produced by these two novel fungi is an object of our further biochemical exploration.

Figure 1 .
Figure 1.(a) Phylogenetic tree of Keratinophyton and Aphanoascus strains based on ITS sequences.The new taxa of Keratinophyton are compared with available sequences of the other related species together with their conidium size, presence of intercalary conidia and ability to grow at 37 °C.Empty triangles and squares represent the absence of the characteristics.(b) Phylogenetic tree based on combination of ITS and LSU sequences for the new taxa of Keratinophyton together with available sequences of the other related species.Numbers at nodes indicate Bayesian probability/ maximum likelihood bootstrap values (≥ 60%).New species are shown in bold.Ctenomyces serratus was used as outgroup.A sequence for K. multiporum was not available for the study.T, ex-type culture.

Table 2 .
Comparison of the key phenotypic characteristics of the phylogenetically close related Keratinophyton spp. a Forming sexual morph (ascospores) in vitro.b Intercalary conidia observed only very rarely.c Intercalary conidia abundant, solitary or in chains.d Intercalary conidia present in older cultures.